Disrupting virulence: in silico discovery of MexL inhibitors to block pyocyanin biosynthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen known for its intrinsic antibiotic resistance and ability to form biofilms, primarily regulated by the redox-active compound, pyocyanin. The transcriptional activator (Multidrug efflux regulator L) MexL plays a crucial role in the biosynthesis of pyocyanin by upregulating key genes such as phz1, phz2, and phzM. Targeting MexL may serve as a novel anti-virulence strategy by suppressing pyocyanin production and attenuating pathogenicity of P. aeruginosa. This study aimed to identify potential inhibitors of MexL using in silico approaches to disrupt its regulatory function and reduce pyocyanin synthesis. The findings suggest that the selected compounds could serve as effective inhibitors of MexL, offering a promising strategy to suppress pyocyanin biosynthesis and reduce P. aeruginosa virulence. For this purpose, molecular docking was conducted between the binding pocket of the MexL protein and compounds selected based on pharmacokinetic properties from the FooDB, NPASS, and LOTUS databases. Furthermore, molecular dynamics simulations were performed on the selected MexL-ligand complexes to evaluate the stability of the protein-ligand complex within a simulated solvated environment under defined conditions. The virtual screening by molecular docking identified four top-scoring ligands FDB000380, LTS0012415, and NPC253096 and ZINC14780782 with binding energies of -8.6, -7.5, -7.1, and -8.8 kcal/mol respectively, showing strong affinities toward the MexL binding pocket. These ligands demonstrated key molecular interactions with residues critical for regulatory function of MexL protein. Molecular dynamics simulations (300 ns) of the three lead MexL-ligand complexes (FDB000380, LTS0012415, and NPC253096) revealed exceptional structural stability, with low backbone RMSD values (0.240-0.309 nm), minimal residue fluctuations, stable radius of gyration, and consistent solvent-accessible surface area profiles. Persistent protein-ligand hydrogen bond networks (occupancy 72.1-100%) confirmed stable polar interactions throughout the trajectories. MM-GBSA and MM-PBSA binding free energy calculations demonstrated superior affinities over the endogenous ligand pyocyanin (ΔG =  - 15.30 to - 21.75 kcal/mol versus - 12.57 kcal/mol). Principal component analysis further revealed restricted conformational dynamics and compound-specific free energy landscapes, collectively supporting these three compounds as thermodynamically stable lead inhibitors of MexL-mediated pyocyanin biosynthesis in Pseudomonas aeruginosa.